Objective The method of metabonomics based on nuclear magnetic resonance (NMR) imaging was used to explore the difference in metabolites of serum and bile, and to analyze the metabolic variation related to the pathogenesis of gallbladder stones between normal people/liver transplantation donors and patients with gallbladder stones. Methods Prospectively collected the serum samples (17 cases) and bile samples (19 cases) in 19 patients with gallbladder stones who underwent surgery in West China Hospital form March 2016 to December 2016, as well as the serum samples of 10 healthy persons and the bile samples of 15 liver transplantation donors at the same time period. The differences of metabolites in the blood and bile in these 3 groups were compared by using 1H-NMR metabonomics technology and chemometric methods. Results The concentrations of valine, alanine, lysine, glutamine, glutamate, pyruvate, creatinine, choline, alpha-glucose, beta-glucose, tyrosine, histidine, and hypoxanthine in serum of patients with gallbladder stones decreased significantly, comparing with those of healthy people without gallbladder stones (P<0.05), while 1, 2-propanediol, acetoacetate, and lactate increased significantly in the serum of patients with gallbladder stones (P<0.05). The concentrations of taurine conjugated bile acids, glycine conjugated bile acids, choline, and phosphatidylcholine decreased significantly in the bile of patients with gallbladder stones when compared with those of liver transplantation donors (P<0.05), while cholesterol increased significantly in the bile of patients with gallbladder stones (P<0.05). Conclusions There are significant differences of the serum and bile metabolites between patients with gallbladder stones and healthy men without gallbladder stones/liver transplantation donors. 1H-NMR metabonomics is helpful to investigate the pathogenesis of gallbladder stones.